Preparation And In Vitro Experimental Study Of An Intervertebral Disc Injectable Platelet-Rich Plasma/Ferulic Acid Hydrogel

Objective: This study aims to prepare a temperature-sensitive injectable hydrogel complex loaded with platelet-rich plasma and ferulic acid,and co-culture it with nucleus pulpocytes in vitro to detect its cytotoxicity and biocompatibility,laying a foundation for subsequent animal tests and clinical trials.Methods: 1.A hydrogel complex was prepared by using hyaluronic acid(HA),ferulic acid(FA),rat platelet-rich plasma(PRP)and batroxase(BTX).The temperature of the hydrogel complex during solidification was determined by stirring method,the structure of the hydrogel complex was scanned by electron microscope,and the drug release of the hydrogel complex was measured in vitro.2.rat nucleus pulposus cells were taken for in vitro culture,and identified after subculture.After being identified as rat nucleus pulposus cells,they were co-cultured with hydrogel complex and then stained for nucleus pulposus cells alive and dead,respectively,to detect the toxicity of hydrogel to nucleus pulposus cells.MTT assay was used to detect the proliferation promotion effect of hydrogel complex on nucleus pulpocytes.Finally,Western Blot and qRT-PCR were used to detect whether the hydrogel complex could promote the proliferation of nucleus pulposa cells at protein level and mRNA level.Results: 1.The freshly prepared hydrogel/platelet-rich plasma/ferulic acid complex was light yellow gel and had certain fluidity.After three repeated experiments,the gel temperatures of hydrogel/platelet-rich plasma/ferulic acid complex were 32℃,33℃ and 33℃,respectively.This indicates that the hydrogel prepared in this experiment can be solidified into solid state at 33℃.The internal structure of the hydrogel/platelet-rich plasma/ferulic acid complex was loose,and the pore diameters of the structures were different,in which a large number of platelets were attached to the gel.Ferulic acid concentrations of hydrogel /PRP/FA,hydrogel /FA and PRP/FA all increased fastest at 12 h,indicating that the drug was released fastest at 12 h.The hydrogel /PRP/FA concentration was the highest.2.Nucleus pulposus cells were successfully cultured in vitro and identified by fluorescence staining.The hydrogel complex was co-cultured with cultured nucleus pulposus cells,and no toxic effect of the hydrogel complex on nucleus pulposus cells was determined by living and dead cell staining.MTT assay was used to detect that the hydrogel complex could promote the proliferation of nucleus pulposa cells.Western Blot and QRT-PCR confirmed that hydrogel complex could promote the proliferation of nucleus pulposa cells at protein level and mRNA level.Conclusion: An injectable thermosensitive hydrogel /PRP/FA complex was successfully prepared,and it was confirmed that the hydrogel complex could promote the proliferation of nucleus pulposus cells by co-culture with the hydrogel complex.